Modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase

Abstract

We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.

Collagen Degradation in the Abdominal Aneurysm

“…16,27 Moreover, the possible involvement of increased collagenase activity 13,14 as the underlying cause of rupture has not been addressed in detail.…”

“…We used specific immunocapture-protease activity assays to validate the MMP mRNA data. These activity assays have been shown to allow quantification of active proteases 16 and, after in vitro activation of the latent MMPs, assessment of the pool of pro-MMP. 16 Direct assessment of MMP collagenases (ie, active enzymes) did not reveal detectable protease activity in the tissue homogenates (all activities were below the detection threshold of the assay).…”

“…These activity assays have been shown to allow quantification of active proteases 16 and, after in vitro activation of the latent MMPs, assessment of the pool of pro-MMP. 16 Direct assessment of MMP collagenases (ie, active enzymes) did not reveal detectable protease activity in the tissue homogenates (all activities were below the detection threshold of the assay). Although this finding may indicate that all collagenases present are in their inactive, latent form, it most likely reflects a technical limitation when assessing protease activity in complex biological samples such as tissue homogenates.…”

“…Harrison, School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK). 16 Conjugated biotinylated antigoat or rabbit anti-IgG were used as secondary antibodies. Sections were stained with Nova Red (Vector Laboratories, Burlingame, CA) and counterstained with Mayer hematoxylin.…”

“…Because this approach does not provide information on the post-transcriptional regulation of protease activity, we quantified tissue expression of specific inhibitors of proteases activity and applied specific protease activity assays and Western blot analyses to address the post-translational regulation of protease activity. 16,17 Data from this study characterize members of the cysteine protease family, cathepsin K, L, and S, along with neutrophil collagenase (MMP-8), as the primary collagenases in AAA and ruptured AAA.…”